1. Field of the Invention
This invention relates to the field of diagnostic assays and the collection and processing of samples therefor.
The ability to measure quantitatively a wide variety of physiologically active compounds, both naturally occurring and synthetic, has become of increasing importance, both as an adjunct to diagnosis and therapy. The medical industry has become increasingly dependent upon the ability to measure various entities in physiological fluids in order to be able to determine the health status of an individual, dosage level for drugs, use of illegal drugs, genomic sequences and the like. Thus, the capability of taking a physiological sample and rapidly analyzing for a particular component has made medical therapies more efficient and increasingly successful.
For the most part diagnostic assays of physiological fluids or biological samples for one or more analytes have required clinical laboratory determinations although there has been an increasing focus on being able to carry out assay determinations in the doctor's office and in the home. Numerous systems have been developed in efforts to try to address the various problems associated with analyses carried out in the clinical laboratory.
There is substantial interest in providing for protocols and devices which are simple, easy to manipulate, and reduce the opportunity for operator failure. The ideal situation would be collection of an unmeasured sample in a container, which is then sealed. Subsequently, the sample could then be introduced into an assay device without opening the sealed container and without the need for accurately measuring the sample. The device into which the sample is introduced provides for precise measurement of the sample to be analyzed, which is important in obtaining a quantitative result.
In may instances blood is a source of a sample to diagnose a patient's health or to monitor the efficacy of drugs that have been administered to the patient. Blood as a source for the determination of these parameters has many deficiencies when used directly or even when diluted with buffer. These deficiencies include: rapid coagulation, the presence of a large number of light absorbing and fluorescent substances, variations in composition, susceptibility to changes in relation to reagents used in assays, and variations In the presence or absence of oxygen. These properties complicate the use of blood as a sample for diagnostic purposes. Various techniques have been employed to avoid these problems, e.g., high dilution, addition of anticoagulants, separation of blood into plasma and its cellular components, and the like. During such manipulations great care must be taken to avoid lysis of red blood cells to avoid the release of hemoglobin, which can interfere with diagnostic assays. Despite the problems associated with the use of blood as the sample medium, in many instances, blood is the only source that provides the information of interest. Therefore, identifying ways of using whole blood, while diminishing the interference from its constituents is highly desirable. There is, therefore, substantial interest in devising new approaches for using and manipulating blood for diagnostic purposes.
Thus, the use of whole blood in diagnostic assays is not unusual in the medical field. When the volume of blood needed to perform the test becomes greater than a few drops, a blood collection container such as a vacuum tube or syringe is used. The subsequent delivery of the sample into the assay requires the transfer of blood from the collection container to an assay device. The transfer increases the risk of both hazardous contact to the clinician as well as alteration of the specimen. Also, in some circumstances, it is desirable to preprocess the blood sample such as by removal of cells from whole blood, lysing cells in whole blood, and so forth.
Certain devices are known for the collection of a sample for a qualitative determination of an analyte of interest. As may be appreciated, the considerations for collection of a sample for a quantitative determination of an analyte are much different. In general, for a qualitative result the collection of a precise amount of a sample is not a consideration.
One area of particular interest in analyses employing whole blood samples is the assessment of platelet function. The role of platelets in mammalian physiology is extraordinarily diverse, but their primary role is in promoting thrombus formation. In many situations, an evaluation of the ability of blood to clot is desired, a parameter that is frequently controlled by the ability of platelets to adhere and/or aggregate. Thus, one may wish to assess the adhesive functions of platelets. For example, one may wish to know whether to administer drugs that will block, or promote, clot formation, or one may need to detect deficiencies in platelet function prior to surgical procedures. In other instances one may be interested in evaluating the effectiveness of a platelet inhibitor that is being tested as a new drug or is being used as approved clinical treatment in a patient.
Platelets are known to aggregate under a variety of conditions and in the presence of a number of different reagents. Platelet aggregation is a term used to describe the binding of platelets to one another. The phenomenon can be induced by adding aggregation inducing agents to platelet rich plasma (PRP) or to whole blood. Platelet aggregation in vitro depends upon the ability of platelets to bind fibrinogen to their surfaces after activation by an aggregation inducing agent such as ADP or collagen.
Platelets play a critical role in the maintenance of normal homeostasis. When exposed to a damaged blood vessel, platelets will adhere to exposed sub endothelial matrix. Following the initial adhesion, various factors released at the site of injury such as thrombin, ADP and collagen activate the platelets. Once platelets are activated, a conformational change occurs in the platelet glycoprotein GPIIb/IIIa receptor allowing it to bind fibrinogen and/or von Willebrand factor.
It is this binding of the multivalent fibrinogen and/or von Willebrand factor molecules by GPIIb/IIIa receptors on adjacent platelets that results in the recruitment of additional platelets to the site of injury and their aggregation to form a hemostatic plug or thrombus.
The success of aspirin, and more recently ticlopidine, in treating, and preventing ischemic complications of thrombosis has stimulated the search for more potent agents. New agents that block platelet GPIIb/IIIa receptors are being developed for use as antithrombotic agents, including peptides and peptidomimetics, based on the arginineglycine-aspartic acid (RGD) and related cell recognition sequences. A recombinant murine/human chimeric antibody Fab fragment (c7E3Fab, abciximab, ReoPrO.TM.) c7E3 has been approved in Europe and the United States for use as adjunctive therapy in high risk angioplasty. Moreover, one additional trial (EPILOG), was stopped early by the Data and Safety Monitoring Boards because of the greater than expected benefit of 61% reduction in thrombotic events with c7E3 in the full range of patients undergoing coronary angioplasty. However, the benefit of GPIIb/IIIa blockers has been accompanied by an increased risk of bleeding. A number of other agents are currently in early and advanced trials, including agents that are orally active.
Intrinsic differences in pharmacokinetics and pharmacodynamics among the GPIIb/IIIa antagonists may affect the dose required to achieve a safe therapeutic antiplatelet effect. Beyond these overall differences in the drugs, however, interindividual variations in drug excretion and metabolism may have an impact on optimal drug dosing. Prolonged therapy, either with parenteral or oral agents, is likely to magnify the importance of interindividual differences, especially with pro drugs and low molecular weight agents that rely on renal excretion or hepatic metabolism.
Direct measurement of GPIIb/IIIa receptor blockade has been reported for only a few GPIIb/IIIa antagonists. An assay to measure GPIIb/IIIa receptor blockade by c7E3 Fab, based upon inhibition of platelet binding of radiolabelled c7E3 has been used to correlate GPIIb/IIIa receptor blockade, inhibition of platelet aggregation, prolongation of the bleeding time, and antithrombotic efficacy in animal models. Based on these results, the target level for coronary artery angioplasty was defined as &gt;80% GPIIb/IIIa receptor blockade, and this level of blockade has proved efficacious in three separate Phase III studies in humans.
Available assays for evaluating GPIIb/IIIa receptor blockade, including platelet aggregation, bleeding time, thromboelastography, clot retraction, radiolabelled antibody binding, and flow cytometry are time consuming, require standardization, or require specialized equipment.
In vitro platelet aggregation is the laboratory method used to assess the in vivo ability of platelets to form the aggregates leading to a primary hemostatic plug. In this technique an aggregating agent such as ADP or collagen is added to whole blood or PRP and aggregation of platelets monitored. Platelet aggregometry is a diagnostic tool that can provide insights difficult to obtain by other techniques, thus aiding in patient diagnosis and selection of therapy. Current methods of monitoring platelet aggregation require expensive, laboratory dedicated instruments that are not easily portable and require standardization to ensure accurate quantitative results. In addition, unless performed using whole blood, results are unlikely to be available for several hours.
Currently there are two detection methods used in instruments with FDA clearance for performing platelet aggregometry: optical and impedance measurements. Optical detection of platelet aggregation is based on the observation that, as platelets aggregate into large clumps, there is an increase in light transmittance. Different aggregation-inducing agents stimulate different pathways of activation and different patterns of aggregation are observed. The main drawback of the optical method is that it must be performed on PRP, necessitating the separation of platelets from red blood cells and adjustment of the platelet count to a standardized value.
Impedance detection can be used to test anti coagulated blood with no need to isolate platelets from other components of the blood, although in many cases the sample is diluted before testing. The method detects aggregation by passing a very small electric current between two electrodes immersed in a sample of blood (or PRP) and measuring electrical impedance between the electrodes. During initial contact with the blood or PRP, the electrodes become coated with a monolayer of platelets. If no aggregating agent is added, no further interactions occur between the platelets and the electrodes and electrical impedance remains constant. When an aggregation inducing agent is added, platelets aggregate on the electrodes and there is an increase in impedance.
The CHRONO LOG Model 530 and Model 540 use the optical method for PRP and the impedance method for whole blood aggregometry. The impedance method has been shown to be substantially equivalent to the optical method for measuring platelet aggregation in PRP.
Various photometers are commercially available for measuring the light absorbance of liquid samples in microtitration plates or other sample holding vessels. Examples of such equipment are the MR 600 Microplate Reader (Dynatech Laboratories, Inc., Alexandria, Va.), and the Vmax Kinetic Microplate Reader (Molecular Devices, Palo Alto, Calif.).
It is desirable to have a rapid and simple platelet function assay including the ability to transfer blood to be tested from a collection container to an assay device without opening the collection container. In the setting of coronary angioplasty, it is desirable to have a platelet aggregation assay that could be conducted at the same time as the activated clotting time (ACT), which is performed to assess the adequacy of heparinization. During chronic infusions of GPIIb/IIIa antagonism, or with chronic oral therapy, periodic monitoring may also be desirable. In certain circumstances, as for example, prior to surgery or an invasive procedure, it may be desirable to rapidly determine whether the effect of the GPIIb/IIIa antagonist has worn off sufficiently to allow the surgery or procedure to be performed without further interventions to reverse the effect of the GPIIb/IIIa inhibitor. Finally, in the event of bleeding complications, a rapid measure of platelet function may be helpful in determining whether the bleeding is due to a high or toxic level of platelet inhibition. The level of platelet inhibition may also be helpful in guiding whether to reverse the drug effect with platelet transfusions or look for other causes of bleeding.
2. Previous Disclosures
O'Brien, J. R., Nature 202:1188 (1964) discloses aggregation studies of 2 mL plasma samples placed in a cuvette in an EEL titrometer or electrophotometer. Each sample is treated individually and aggregation is said to occur when the optical transmission increases.
Mills, D.C.B., and Roberts, G.C.K., J. Physiol. 193:443 453 (1967) disclose platelet aggregation measurements in a modified EEL Long Cell Absorptiometer (Evans Electroselenium Ltd., Halstead, Essex, U.K.). The measurements are taken on a 1 mL plasma sample, stirred from below by a magnetic stirrer while continuous recordings are made.
Michal, F., and Born, G.V.R., Nature New Biol. 231 220 (1971) disclose a modification of the traditional optical method of measuring aggregation which permits the simultaneous measurement of scattered and transmitted light. This method encompasses a modification of the cuvette chamber of an aggregometer to allow for the measurement of light scattered at right angles to the incident light beam. In the aggregometer the incident light illuminates a suspension of platelets which are kept in rapid motion by a magnet rotating in the bottom of the glass tube at 1,000 rpm. The sample volume is about 1 mL and the optical density is read individually for each sample which is kept in a water-jacketed environment at 37.degree. C.
Fratontoni, et al., in U.S. Pat. No. 5,325,925 disclose the use of an agitated microtiter plate to assess aggregation in PRP.
Shaw, et al., in U.S. Pat. No. 5,427,913 disclose a method for determining platelet function in PRP by contacting the platelets in suspension with an immobilized extracellular matrix protein while applying mechanical stimulus to the platelets, and determining the platelet activation produced by various indicia.
Coagulation monitors are known for the analysis of whole blood. U.S. Pat. No. 3,695,842 describes a method of analyzing the transformation of a liquid, e.g. blood, to a gelatinous or solid mass. The coagulation system uses a vacutainer with all the necessary reagents, as well as a ferromagnetic component. Once the blood sample has been drawn into the vacutainer, it is placed into the instrument in an inclined manner. This procedure makes the ferromagnetic component sit at the bottom of the tube in close proximity to a magnetic reed switch. As the sample is rotated, gravity ensures that the component remains close to the reed switch. However, as the blood starts to clot, viscosity increases to the point where the component starts to rotate with the blood sample. The reed switch is thus activated, enabling a coagulation time to be estimated.
Hillman, et al., disclose a unit use cartridge in which dry reagents are placed into the analyzer which is then heated to 37.degree. C. before a drop of brood is introduced. The sample is mixed with the reagent by capillary draw. The detection mechanism is based on laser light passing through the sample. Blood cells moving along the flow path yield a speckled pattern specific to unclotted blood. When the blood clots, movement ceases producing a pattern specific to clotted blood. Several patents disclose aspects of this technology and are described further below.
U.S. Pat. No. 4,756,884 discloses the component parts of the cartridge technology, which is based on capillary draw, including certain antibodies and reagents for blood clotting.
U.S. Pat. No. 4,948,961 discloses the components and method of use of an optical simulator cartridge used with the above instrument. U.S. Pat. No. 4,963,498 discloses a method of obtaining chemical information from the capillary draw cartridge. U.S. Pat. No. 5,004,923 discloses optical features by which the above instrument interrogates the cartridge
Shenaq and Saleem, in "Effective Hemostasis in Cardiac Surgery," Eds: Ellison, N. and Jobes, D. R., Saunder & Co. (1988), utilize a sonic probe that is inserted into a cuvette containing the sample and reagents. The sonic probe responds to clot formation in the cuvette and, thus, can be used to measure the coagulation time.
PCT application WO 89/06803 describes a unit use cartridge having two capillary tubes that simultaneously draw blood from a single finger stick. The design allows for duplicate measurement or two different measurements based on different reagent coatings. Blood coagulation is measured by charges in light permeability through the capillary tube.
U.S. Pat. No. 5,110,727 describes another format based on the use of magnetic particles mixed into a dry reagent contained within a flat capillary chamber. An applied oscillating magnetic field from the instrument causes the particles to oscillate once the reagent has dissolved in the blood. This motion is monitored optically. When the blood clots, the particles become entrapped and motion is diminished. Fibrinolysis assays are performed by monitoring the reverse process (See, Oberhardt, et al., Clin. Chem. (1992) 37:520).
Machado, et al., J. Acoust. Soc. Am (1991) 90:1749 describe another method of detecting coagulation based on ultrasonic scattering from 200 micron glass spheres suspended in a blood sample. Amplitude and phase changes of the scattered waves are used to detect coagulation.
Varon, et al., in U.S. Pat. No. 5,523,238, describe a method for determining platelet function by introducing a blood sample into a vessel having a flat bottom, the inner surface of which is covered with a substrate capable of inducing platelet adhesion thereto and aggregation; rotating the preparation inside the vessel, inducing shear forces at the surface which aggregate the platelets; and determining parameters of the adhered blood platelets, such as number of adhered platelets, aggregate size, aggregate morphology, total area covered by the aggregates, and distribution of adhered platelets or aggregates on the surface.
Coller, in pending U.S. patent applications Ser. Nos. 08/315,026 and 08/754,773, describes a method for the analysis of whole blood for GPIIb/IIIa receptor activity. The disclosed method relies upon the visual observation of platelet mediated agglutination of fibrinogen coated beads.
The disclosure of all the aforementioned patents and publications is incorporated herein by reference.